Michele Pamela Calos

My research over the course of my career has focused
on creating and utilizing innovative genetic systems
involving chromosomes and plasmids. The body of work
has encompassed studies elucidating gene expression,
transposable elements, mutation and repair, DNA
replication, DNA recombination, genomic integration,
gene therapy, and stem cell therapy.

This review concentrates on my work over the past
ten years, which has focused on creating
sequence-specific integration systems and using
them, predominantly to advance the science and
clinical application of gene therapy and stem cell
therapy. I will briefly recap my earlier research,
which laid the groundwork for the present activities
and demonstrates my general approach.

Earlier research

At Oxford, I received a broad undergraduate
education in biology, ranging from biochemistry and
biophysics to genetics and evolution. As a graduate
student with Nobel laureate Prof. Walter Gilbert at
Harvard, I took advantage of newly available cloning
and sequencing technology to be the first to clone
the classic lac repressor gene, determine
the DNA sequence of its promoter, and determine the
DNA sequences at the junctions between transposable
elements and their chromosomal targets. During a
brief postdoc in Jeffrey Miller's lab in Geneva,
Switzerland, I made the transition from working with
E. coli to mammalian cells, creating a
forerunner of the first shuttle vector system for
analyzing mutation in human cells.

I obtained a Stanford Assistant Professor position
at this time and began a program in human genetics
utilizing the innovative shuttle vector approach. I
established a laboratory in the Department of
Genetics, where we were able to demonstrate the
utility of the shuttle vector approach for the
analysis of mutation in mammalian cells. This
approach soon made its mark on the mutation and
repair field and provided the first sequence-level
analysis of mutation in mammalian cells that was of
sufficient power to define the mutational spectra
produced by a variety of mutagens.

I was fascinated by the chromosome-like behavior of
the shuttle vectors developed by my lab. We studied
plasmids based on Epstein-Barr virus and from these
EBV vectors, my lab developed the first autonomous
replication system for mammalian cells that used
human genomic sequences as replication origins. The
data we produced indicated that mammalian cells
lacked the precisely defined replication origins of
viruses and prokaryotes, and instead used more
diffuse signals spread over a large area. This view
was controversial in the field, but has stood the
test of time and is now accepted as correct.

The field of gene therapy was developing during this
period, based largely on the use of viral vectors
for introduction of DNA into cells. I realized that
the vectors my lab had created to study replication,
with their ability to replicate and be retained in
mammalian cells, might represent a viable
alternative that could help solve the problem of
transient expression plaguing non-viral approaches
to gene therapy that used conventional plasmid DNA.
We thus made the transition into the gene therapy
field. The extrachromosomal vectors we developed
were successful in numerous animal studies and have
provided curative levels of therapeutic proteins
such as a1 anti-trypsin and factor IX. On the other
hand, the extrachromosomal vectors had the drawbacks
for gene therapy of being not completely stable in
dividing cells and of requiring the expression of a
viral protein, EBNA1.

Site-specific integration

To overcome these limitations, we sought to create
an integrating vector system. Integration offers the
most reliable route to stability, because a vector
then acquires the impressive stability of the
chromosomes. This feature is critical in dividing
cells, such as stem cells. Moving beyond previous
gene therapy vectors, which integrate randomly, we
became committed to creating sequence-specific
integration systems. We saw the need for such
technology, not only for gene therapy, but also for
creation of cell lines, transgenic organisms,
protein production, and many other applications.
Thus, my lab accepted the challenge of creating such
technology, which had previously been largely
missing from the genetics toolkit.

Recombinant DNA technology of the 1970s provided the
methodology to manipulate DNA molecules precisely in
a test tube. However, the field of genetics had been
largely unable to create precise and efficient
genomic integration in living cells of higher
organisms. The contribution of methodologies that
would permit efficient integration at native
sequences in the chromosomes became a central goal
of my lab's research program.

The solution we devised to achieve sequence-specific
integration was original and relied on use of
microbial site-specific recombinases that in theory
would recognize sequences in mammalian chromosomes
having degenerate homology to their actual
recognition sites. This hypothesis, based on the
statistics of large genomes and the nature of
protein-DNA interactions, proved to be correct. We
pioneered this idea using the prokaryotic resolvase
Cre, demonstrating for the first time that the
enzyme can recognize "pseudo" lox sequences
in the human and mouse genomes that depart
significantly from the native 34-bp loxP
site that Cre normally recognizes. However, Cre was
not a good enzyme for integration, because it also
performed excision efficiently, which reversed
integration events.

We found a complete solution to this problem by
using a different recombinase, the phage φC31
integrase. This enzyme from a Streptomyces
phage could perform unidirectional recombination at
its attachment (att) sites without host
co-factors. We recognized that these were the
properties needed to create a useful integration
tool and were the first to use φC31 integrase in
eukaryotic cells. My lab rapidly and successfully
constructed expression and assay systems for φC31
integrase and demonstrated the ability of the enzyme
to function on extrachromosomal plasmids in human
cells in a landmark paper in 2000 (Groth et al.,
2000).

We extended this study the next year with the first
demonstration that the φC31 integrase could indeed
find native sequences in the human and mouse genomes
where it could carry out site-specific integration
(Thyagarajan et al, 2001). The enzyme recognized a
number of "pseudo" att sites in the human
genome that had partial identity to attP.
Integration generally occurred at a single copy per
cell. In order to characterize the chromosomal
sequences where integration took place, we carried
out a large study of integration sites in human cell
lines (Chalberg et al., 2006). The study revealed a
28-bp consensus sequence present at the sites of
integration and a hierarchy of preferred integration
sites in the genome having sequences related to this
consensus. Chromatin context features also played a
role in creating favorable integration sites.
Statistical analysis suggested that φC31 integrase
could potentially integrate at approximately 370
genomic locations in human cell lines. It is
probable, based on our studies in animals summarized
below, that a considerably narrower set of target
sites is actually available in vivo, which
provides greater site-specificity.

This level of site-specificity was an improvement of
several orders of magnitude over the quasi-random
integration exhibited by previously available
integrating vectors, such as those based on
oncoretroviruses, lentiviruses, and transposases.
The tighter specificity of φC31 integrase
substantially reduced the insertional mutagenesis
risk during gene therapy. Control over integration
location is considered critically important,
especially in stem cell populations such as
hematopoietic stem cells. The need for site-specific
integration was illustrated by a gene therapy
clinical trial in Paris. Leukemia occurred in
several patients as a result of expression of an
oncogene, activated by random integration of the
retroviral gene therapy vector.

Research tools

As research tools, our site-specific integration
systems are of widespread utility. One area of
intense activity is use of φC31 integrase for
generation of transgenic organisms. We initiated
this research area by demonstrating the utility of
our system for generating transgenic Drosophila
in a collaboration with Roel Nusse of the Dept. of
Developmental Biology. By injecting φC31 integrase
mRNA into fly embryos, we catalyzed genomic
integration of an injected attB plasmid,
producing transgenic flies at an unprecedented level
of efficiency (50%) and precision (100%) (Groth et
al., 2004). This methodology has been widely adopted
by the Drosophila research community.

The φC31 system has also found utility in the
community of researchers seeking to modify the
genome of mosquitoes in order to combat malaria and
other diseases. Success has been achieved as well by
using φC31 integrase to generate transgenic Xenopus
tadpoles at a frequency of 30-40%. Moreover, φC31
integrase has been used to develop methodologies for
the generation of transgenic zebrafish, chickens,
and other important research and industrial
organisms. We have also demonstrated the utility of
the system for robust protein production in
mammalian cells and for construction of cell lines
with integrations at the same position, which are
useful for the systematic analysis, for example, of
gene control regions in a constant chromosomal
context. These types of achievements demonstrate
that we have created a system of broad utility for
genetics research.

Gene therapy
A key goal of my laboratory is to use the
integration systems we create to cure disease. To
further this goal, we completed several studies in
animal models that illustrated the utility of the
φC31 integrase system for gene therapy.

In our first animal study, we demonstrated that we
could introduce normal levels of human factor IX to
mice by site-specifically integrating in liver a
vector carrying the human factor IX gene. We
co-delivered a factor IX-attB plasmid with a
plasmid encoding the φC31 integrase. DNA was
delivered by high-pressure tail vein injection of
the plasmids, in a manner that directed DNA to the
liver. We observed that the majority of integrations
in hepatocytes occurred at a position on chromosome
2 and that we obtained high levels of factor IX that
would be sufficient to cure hemophilia in patients.
More recently, we carried out a similar study in
disease model mice with factor IX (Keravala et al.,
2011) and factor VIII deficiency (Chavez et al.,
2012).

We also demonstrated the utility of the
site-specific integration method in many other gene
therapy applications, including skin, retina,
joints, and neural stem cells. In collaboration with
Tom Rando in Neurology, we showed that integrase
provides greater, long-lasting expression of
dystrophin in skeletal muscle in a mouse model of
muscular dystrophy (Bertoni et al., 2006).

Stem Cell Therapy
Most recently, we have become interested in applying
the power of our genome engineering systems to the
creation and modification of stem cells for
therapeutic uses. For example, we demonstrated the
use of φC31 integrase for reprogramming adult cells
into induced pluripotent stem cells (iPSC; Karow et
al, 2011). After iPSC from patients with genetic
diseases such as muscular dystrophy are corrected
with genetic engineering methods, the corrected
cells can be transplanted back to the patient for
engraftment and tissue repair. Using the latest,
evolving methods for genome engineering, we are
currently pursuing these types of approaches to
develop novel strategies to treat Duchenne and limb
girdle muscular dystrophies.